Date Published: January 13, 2009
Publisher: Public Library of Science
Author(s): Raja K Sivamani, Christine E Pullar, Catherine G Manabat-Hidalgo, David M Rocke, Richard C Carlsen, David G Greenhalgh, R. Rivkah Isseroff, Jeffrey Davidson
Abstract: BackgroundStress, both acute and chronic, can impair cutaneous wound repair, which has previously been mechanistically ascribed to stress-induced elevations of cortisol. Here we aimed to examine an alternate explanation that the stress-induced hormone epinephrine directly impairs keratinocyte motility and wound re-epithelialization. Burn wounds are examined as a prototype of a high-stress, high-epinephrine, wound environment. Because keratinocytes express the β2-adrenergic receptor (β2AR), another study objective was to determine whether β2AR antagonists could block epinephrine effects on healing and improve wound repair.Methods and FindingsMigratory rates of normal human keratinocytes exposed to physiologically relevant levels of epinephrine were measured. To determine the role of the receptor, keratinocytes derived from animals in which the β2AR had been genetically deleted were similarly examined. The rate of healing of burn wounds generated in excised human skin in high and low epinephrine environments was measured. We utilized an in vivo burn wound model in animals with implanted pumps to deliver β2AR active drugs to study how these alter healing in vivo. Immunocytochemistry and immunoblotting were used to examine the up-regulation of catecholamine synthetic enzymes in burned tissue, and immunoassay for epinephrine determined the levels of this catecholamine in affected tissue and in the circulation. When epinephrine levels in the culture medium are elevated to the range found in burn-stressed animals, the migratory rate of both cultured human and murine keratinocytes is impaired (reduced by 76%, 95% confidence interval [CI] 56%–95% in humans, p < 0.001, and by 36%, 95% CI 24%–49% in mice, p = 0.001), and wound re-epithelialization in explanted burned human skin is delayed (by 23%, 95% CI 10%–36%, p = 0.001), as compared to cells or tissues incubated in medium without added epinephrine. This impairment is reversed by β2AR antagonists, is absent in murine keratinocytes that are genetically depleted of the β2AR, and is reproduced by incubation of keratinocytes with other β2AR-specific agonists. Activation of the β2AR in cultured keratinocytes signals the down-regulation of the AKT pathway, accompanied by a stabilization of the actin cytoskeleton and an increase in focal adhesion formation, resulting in a nonmigratory phenotype. Burn wound injury in excised human skin also rapidly up-regulates the intra-epithelial expression of the epinephrine synthesizing enzyme phenylethanolamine-N-methyltransferase, and tissue levels of epinephrine rise dramatically (15-fold) in the burn wounded tissue (values of epinephrine expressed as pg/ug protein ± standard error of the mean: unburned control, 0.6 ± 0.36; immediately postburn, 9.6 ± 1.58; 2 h postburn, 3.1 ± 1.08; 24 h post-burn, 6.7 ± 0.94). Finally, using an animal burn wound model (20% body surface in mice), we found that systemic treatment with βAR antagonists results in a significant increase (44%, 95% CI 27%–61%, p < 0.00000001) in the rate of burn wound re-epithelialization.ConclusionsThis work demonstrates an alternate pathway by which stress can impair healing: by stress-induced elevation of epinephrine levels resulting in activation of the keratinocyte β2AR and the impairment of cell motility and wound re-epithelialization. Furthermore, since the burn wound locally generates epinephrine in response to wounding, epinephrine levels are locally, as well as systemically, elevated, and wound healing is impacted by these dual mechanisms. Treatment with beta adrenergic antagonists significantly improves the rate of burn wound re-epithelialization. This work suggests that specific β2AR antagonists may be apt, near-term translational therapeutic targets for enhancing burn wound healing, and may provide a novel, low-cost, safe approach to improving skin wound repair in the stressed individual.
Partial Text: Major burn injury, characterized by burns of greater than 20% of body surface area, is associated with significant multisystem dysfunction . The absence of a protective epithelium in the burn wound results in the comorbidities of fluid and electrolyte loss and wound infection. Despite recent therapeutic advances, the mortality rate from major burns remains high, with a resultant pressing need for the development of economical and widely available therapeutic approaches to enhance the rate of wound re-epithelialization and restoration of the protective epithelial barrier.
To determine if epinephrine could directly modulate keratinocyte motility, we treated cultured human keratinocytes with epinephrine at levels that are comparable to that found in the circulation of burn-wounded patients (10 nM) . Epinephrine decreases both keratinocyte migratory speed (from 1.587 to 0.388, 95% confidence interval [CI] for the difference using Tukey’s HSD 0.891–1.508, p = 0.00001) and in vitro scratch wound closure rates (difference in percent reduction from baseline of −30.11% with 95% CI −33.33% to −26.89%, p < 0.0001), while the addition of a βAR antagonist, timolol, reverses these effects (Figure 1A, migration speed 1.791, 95% CI for the difference between timolol + epinephrine and epinephrine alone 1.070–1.736, p = 0.00001 for cell migration, difference in percent reduction from baseline between timolol+epinephrine and epinephrine alone 34.55% with 95% CI 31.33%–37.77%, p < 0.0001 for in vitro scratch wounding) suggesting that the epinephrine effects are mediated through the βAR. The specific receptor dependence of the observed response was confirmed by examining the migratory rates of keratinocytes isolated from the epidermis of mice in which the β2AR had been genetically deleted . Wild-type β2AR expressing keratinocytes are slowed by the inclusion of 10 nM epinephrine (from a speed of 1.165 to 0.735, 95% CI for the difference 0.28–0.58 by Tukeys's HSD, p = 0.001 ) while the β2AR −/− keratinocytes show no response in migratory rate to either agonist or antagonist (Figure 1B, all differences less than 0.06 with an overall mean of 1.15, none significant). Taken together, these studies demonstrate how stress, with its associated elevation of the stress hormone epinephrine, can impair cutaneous wound healing: by activation of the epidermal keratinocyte β2AR, blunting of promigratory signaling pathways, stabilizing a stationary cell morphologic phenotype, and subsequently diminishing migratory speed required for efficient wound re-epithelialization. This work also expands the developing understanding of how stress can modulate wound healing, which until now has focused primarily on the hypothalamic-pituitary-adrenal (HPA) and glucocorticoid-mediated pathway. The results presented here demonstrate that the neuroendocrine stress response, with the emblematic increase in epinephrine, can also directly affect the wound repair process. Both genetic and pharmacologic approaches demonstrate the dependence of the epinephrine-mediated impairment on an intact keratinocyte β2AR, and implicate this receptor in the impairment of healing in the burn-wound stress model used here. While some of the pathways for glucocorticoid-induced impairment of healing have been elucidated [4,7], the glucocorticoid and catecholamine pathways may crosstalk to impact healing. Here we note a burn-induced rapid rise in epidermal PNMT in the immediate burn wound vicinity. Levels of this epinephrine synthesizing enzyme have been reported to be elevated by stress in multiple tissue by both glucocorticoid-dependent and -independent mechanisms . The PNMT promoter has a glucocorticoid response element , and elevations in circulating cortisol induce rapid increases in the PNMT transcripts and protein expression . Interestingly, epidermal keratinocytes have recently been found to have glucocorticoid synthesizing ability, and this synthesis is increased in wounding [33,34]. Thus, a local hormonal signaling pathway can be envisioned, where wounding induces epidermal glucocorticoid synthesis, which can drive the observed up-regulation of PNMT, and where temporal imbalances in the pathway could result in sustained elevation of both local glucocorticoids and catecholamines, with resultant impairment of healing. Source: http://doi.org/10.1371/journal.pmed.1000012